Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase
Autor: | Brett A. Neilan, Russel Pickford, Sarah E. Ongley, Elke Dittmann, Jan-Christoph Kehr, Angela H. Soeriyadi |
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Rok vydání: | 2021 |
Předmět: |
01 natural sciences
Biochemistry 03 medical and health sciences chemistry.chemical_compound Biosynthesis Valine Nonribosomal peptide Peptide Synthases Lyngbya Toxins Molecular Biology Adenylylation 030304 developmental biology chemistry.chemical_classification 0303 health sciences Molecular Structure 010405 organic chemistry Organic Chemistry Mutagenesis 0104 chemical sciences chemistry Mutagenesis Site-Directed Molecular Medicine Heterologous expression Leucine Isoleucine |
Zdroj: | ChemBioChem. 23 |
ISSN: | 1439-7633 1439-4227 |
Popis: | Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein. |
Databáze: | OpenAIRE |
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